Identification of adenovirus (ad) penton base neutralizing epitopes by use of sera from patients who had received conditionally replicative ad (addl1520) for treatment of liver tumors

Sera from 17 patients with primary and secondary liver tumors who had been administered oncolytic adenovirus (Ad) mutant Addl1520 were analyzed for anti-Ad neutralization titers and antibodies to the Ad major capsid proteins hexon, penton base (Pb), and fiber. The antibodies recognized mainly conformational epitopes in hexon and both linear and conformational epitopes in Pb and fiber. Pb-specific antibodies were isolated from serum samples that had been obtained prior to and during the course of the treatment of four of these patients. We found that the Pb antibodies had a significant contribution toward anti-Ad neutralization, and this mainly occurred at the step of virus internalization. The Pb antigenic epitopes were determined by phage biopanning and were mapped to 10 discrete regions, which made up three major immunodominant domains within residues 51 to 120, 193 to 230, and 311 to 408, respectively. One of these domains (residues 311 to 408) overlapped the highly conserved, integrin-binding RGD (Arg-Gly-Asp) motif. The contribution of antibodies directed to RGD and other epitopes in Ad neutralization activity was determined indirectly by using a phage-mediated depletion assay. Our results suggested that circulating RGD antibodies were not prevalent and were poorly neutralizing and that other peptide motifs within residues 51 to 60, 216 to 226, and 311 to 408 in Pb sequence represented major target sites for neutralizing antibodies.     

Functional and structural basis of carnitine palmitoyltransferase 1A deficiency

Carnitine palmitoyltransferase 1A (CPT1A) is the key regulatory enzyme of hepatic long-chain fatty acid beta-oxidation. Human CPT1A deficiency is characterized by recurrent attacks of hypoketotic hypoglycemia. We presently analyzed at both the functional and structural levels five missense mutations identified in three CPT1A-deficient patients, namely A275T, A414V, Y498C, G709E, and G710E. Heterologous expression in Saccharomyces cerevisiae permitted to validate them as disease-causing mutations. To gain further insights into their deleterious effects, we localized these mutated residues into a three-dimensional structure model of the human CPT1A created from the crystal structure of the mouse carnitine acetyltransferase. This study demonstrated for the first time that disease-causing CPT1A mutations can be divided into two categories depending on whether they affect directly (functional determinant) or indirectly the active site of the enzyme (structural determinant). Mutations A275T, A414V, and Y498C, which exhibit decreased catalytic efficiency, clearly belong to the second class. They are located more than 20 A away from the active site and mostly affect the stability of the protein itself and/or of the enzyme-substrate complex. By contrast, mutations G709E and G710E, which abolish CPT1A activity, belong to the first category. They affect Gly residues that are essential not only for the structure of the hydrophobic core in the catalytic site, but also for the chain-length specificity of CPT isoforms. This study provides novel insights into the functionality of CPT1A that may contribute to the design of drugs for the treatment of lipid disorders.     

Laminin alpha5 chain is required for intestinal smooth muscle development

Laminins (comprised of alpha, beta, and gamma chains) are heterotrimeric glycoproteins integral to all basement membranes. The function of the laminin alpha5 chain in the developing intestine was defined by analysing laminin alpha5(-/-) mutants and by grafting experiments. We show that laminin alpha5 plays a major role in smooth muscle organisation and differentiation, as excessive folding of intestinal loops and delay in the expression of specific markers are observed in laminin alpha5(-/-) mice. In the subepithelial basement membrane, loss of alpha5 expression was paralleled by ectopic or accelerated deposition of laminin alpha2 and alpha4 chains; this may explain why no obvious defects were observed in the villous form and enterocytic differentiation. This compensation process is attributable to mesenchyme-derived molecules as assessed by chick/mouse alpha5(-/-) grafted associations. Lack of the laminin alpha5 chain was accompanied by a decrease in epithelial alpha3beta1 integrin receptor expression adjacent to the epithelial basement membrane and of Lutheran blood group glycoprotein in the smooth muscle cells, indicating that these receptors are likely mediating interactions with laminin alpha5-containing molecules. Taken together, the data indicate that the laminin alpha5 chain is essential for normal development of the intestinal smooth muscle and point to possible mesenchyme-derived compensation to promote normal intestinal morphogenesis when laminin alpha5 is absent.     

parachute/n-cadherin is required for morphogenesis and maintained integrity of the zebrafish neural tube

N-cadherin (Ncad) is a classical cadherin that is implicated in several aspects of vertebrate embryonic development, including somitogenesis, heart morphogenesis, neural tube formation and establishment of left-right asymmetry. However, genetic in vivo analyses of its role during neural development have been rather limited. We report the isolation and characterization of the zebrafish parachute (pac) mutations. By mapping and candidate gene analysis, we demonstrate that pac corresponds to a zebrafish n-cadherin (ncad) homolog. Three mutant alleles were sequenced and each is likely to encode a non-functional Ncad protein. All result in a similar neural tube phenotype that is most prominent in the midbrain, hindbrain and the posterior spinal cord. Neuroectodermal cell adhesion is altered, and convergent cell movements during neurulation are severely compromised. In addition, many neurons become progressively displaced along the dorsoventral and the anteroposterior axes. At the cellular level, loss of Ncad affects beta-catenin stabilization/localization and causes mispositioned and increased mitoses in the dorsal midbrain and hindbrain, a phenotype later correlated with enhanced apoptosis and the appearance of ectopic neurons in these areas. Our results thus highlight novel and crucial in vivo roles for Ncad in the control of cell convergence, maintenance of neuronal positioning and dorsal cell proliferation during vertebrate neural tube development.     

Cloning of two tryptophan hydroxylase genes expressed in the diencephalon of the developing zebrafish brain

The monoamine serotonin (5-HT) exerts key neuromodulatory activities in all animal phyla, but the development and function of the serotonergic system is still incompletely understood. The zebrafish Danio rerio is an excellent model to approach this question since it is amenable to a combination of genetic, molecular and embryological studies. In order to characterize the organization of serotonergic neurons in the zebrafish we cloned two cDNAs encoding distinct forms of tryptophan hydroxylase (Tph), the rate-limiting enzyme in serotonin synthesis. We report here the pattern of expression of these two genes in relation with immunoreactive TH and 5-HT nuclei in the developing zebrafish embryo and early larva. tphD1 expression starts at 22 h post-fertilization (hpf) in the epiphysis and in basal spinal cells. Expression persists in the epiphysis until at least 4 days (dpf). Between 48 hpf and 3 dpf, tphD1 expression is initiated in retinal amacrine cells and in restricted preoptic and posterior tubercular nuclei within the basal diencephalon. At 3 and 4 dpf, tphD1 expression is newly initiated in the caudal hypothalamus and in branchial arches-associated neurons. tphD2 mRNA is detected transiently (between 30 somites and 32 hpf) in a restricted preoptic nucleus. All sites of tphD1 or D2 expression within the anterior central nervous system are also immunoreactive for 5-HT, but are not positive for TH. However, neither tphD gene is expressed in raphe nuclei, suggesting that additional tph gene(s) exist in zebrafish to account for 5-HT synthesis in that location. The co-expression of tphD1, tphD2 and 5-HT in the zebrafish diencephalon appears in striking contrast to the situation in mammals, where diencephalic serotonin results from re-uptake rather than from local production.     

Aminoglycoside-derived cationic lipids as efficient vectors for gene transfection in vitro and in vivo

Background

Cationic lipids are at present very actively investigated for gene transfer studies and gene therapy applications. Basically, they rely on the formation of DNA/lipid aggregates via electrostatic interactions between their cationic headgroup and the negatively charged DNA. Although their structure/activity relationships are not well understood, it is generally agreed that the nature of the positive headgroup impacts on their transfection activity. Thus, we have directed our efforts toward the development of cationic lipids with novel cationic moieties. In the present work, we have explored the transfection potential of the lipophilic derivatives of the aminoglycoside kanamycin A. Indeed, aminoglycosides, which are natural polyamines known to bind to nucleic acids, provide a favorable scaffold for the synthesis of a variety of cationic lipids because of their structural features and multifunctional nature.

Methods and results

We report here the synthesis of a cationic cholesterol derivative characterized by a kanamycin A headgroup and of its polyguanidinylated derivative. The amino‐sugar‐based cationic lipid is highly efficient for gene transfection into a variety of mammalian cell lines when used either alone or as a liposomal formulation with the neutral phospholipid dioleoylphosphatidylethanolamine (DOPE). Its polyguanidinylated derivative was also found to mediate in vitro gene transfection. In addition, colloidally stable kanamycin‐cholesterol/DOPE lipoplexes were found to be efficient for gene transfection into the mouse airways in vivo.

Conclusions

These results reveal the usefulness of cationic lipids characterized by headgroups composed of an aminoglycoside or its guanidinylated derivative for gene transfection in vitro and in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

Genetic diversity and molecular detection of three toxic dinoflagellate genera (Alexandrium,Dinophysis, and Karenia) from French coasts

The objectives of this study were 1) to study the genetic diversity of the Alexandrium, Dinophysis and Karenia genera along the French coasts in order to design probes targeting specific DNA regions, and 2) to apply PCR-based detection to detect these three toxic dinoflagellate genera in natural samples. Genetic diversity of these toxic taxa was first studied from either cultures or cells isolated from Lugol-fixed field samples. By this way, partial sequences of the large ribosomal subunit (LSU rDNA) including the variable domains D1 and D2 of A. minutum, Alexandrium species inside the tamarensis complex, the D. acuminata complex and K. mikimotoi were obtained. Next, specific primers were designed for a selection of toxic algae and used during semi-nested PCR detection. This method was tested over a 3-month period on water samples from the Bay of Concarneau (Brittany, France) and on sediment from the Antifer harbor (The English Channel, France). Specificity and sensitivity of this molecular detection were evaluated using the occurrence of target taxa reported by the IFREMER (Institut Fran?ais de Recherche pour l'Exploitation de la Mer) monitoring network based on conventional microscopic examination. This work presents the first results obtained on the biogeographical distribution of genotypes of these three toxic genera along the French coasts.     

Transition metal-carbonyl labeling of biotin and avidin for use in solid-phase carbonyl metallo immunoassay (CMIA)

The preparation of several transition metal-carbonyl tracers of biotin and avidin is described. Multiple labeling of avidin was achieved by acylation of some of its amine-bearing residues with N-succinimidyl 4-pentynoate (dicobalt hexacarbonyl). By varying the initial amount of this complex, protein conjugates with the extent of derivatization of up to 13 were obtained. Biotin was labeled with one (eta5-cyclopentadienyl)manganese tricarbonyl moiety by reaction of biotin hydrazide, whereas multiple labeling was reached by successive conjugation of biotin and N-succinimidyl 4-pentynoate (dicobalt hexacarbonyl) or (eta5-cyclopentadienyl)iron dicarbonyl (eta1-N-maleimidato) to poly-L-lysine or fourth generation Starburst dendrimer. All the conjugates displayed a good to excellent bioaffinity toward their respective counterparts, as measured by competitive enzymatic assays.     

Analysis of the Escherichia coli Tol-Pal and TonB systems by periplasmic production of Tol,TonB, colicin,or phage capsid soluble domains

The aim of this review is to describe an in vivo assay of the interactions taking place in the Tol-Pal or TonB-ExbB-ExbD envelope complexes in the periplasm of Escherichia coli and between them and colicins or g3p protein of filamentous bacteriophages. Domains of colicins or periplasmic soluble domains of Tol or TonB proteins can be artificially addressed to the periplasm of bacteria by fusing them to a signal sequence from an exported protein. These domains interact specifically in the periplasm with the Tol or TonB complexes and disturb their function, which can be directly detected by the appearance of specific tol or tonB phenotypes. This technique can be used to detect new interactions, to characterize them biochemically and to map them or to induce tol or tonB phenotypes to study the functions of these two complexes.